Chromosomal DNA isolation from E. coli protocol

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<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>culture grown in your favorite medium</li><li>ice-cold Killing Buffer</li><li>TE</li><li>10% SDS</li><li>0.5M EDTA</li><li>isopropanol</li><li> <a name="RNase A">RNase A <tab><div style="margin-right: 600px;">(25mg/ml)</div></a></li><li> <a name="proteinase K">proteinase K <tab><div style="margin-right: 600px;">(25mg/ml)</div></a></li><li>ethanol</li><li>3M Na-Acetate</li><li>distilled water</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Incubator</li></ul><h2>Steps:</h2><ol><li>Measure out 1 volume ice-cold Killing Buffer into culture grown in your favorite medium. Vortex the mixture for a few secs. Store the tube on ice. Samples should be processed as fast as possible. </li><li>Centrifuge at maximum speed for 3 mins at 4°C, gently aspirate out the supernatant and discard it. </li><li>Measure out 300 µl of TE into Eppendorf tube (1). Add 40 µl of 10% SDS. Add 3 µl of 0.5M EDTA. Resuspend pellet by vortexing/by shaking vigorously. </li><li>Incubate at 65°C for 5 mins. </li><li>Add 750 µl of isopropanol. Vortex the mixture for a few secs. </li><li>Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it. </li><li>Add 500 µl of TE. Add 2 µl of <a href="#RNase A" >RNase A</a>. Resuspend pellet by vortexing/by shaking vigorously. </li><li>Incubate at 65°C for 30 mins. </li><li>Add 2 µl of <a href="#proteinase K" >proteinase K</a>. Incubate at 37°C for 15 mins. </li><li>phenol extract (2x phenol & 2x chlorophorm). </li><li>Add 1 ml of ethanol. Add 40 µl of 3M Na-Acetate. Store at room temperature for 12 hrs(overnight). </li><li>Centrifuge at maximum speed for 15 mins at 4°C, gently aspirate out the supernatant and discard it. Add 50 µl of distilled water. Resuspend pellet by vortexing/by shaking vigorously. </li></ol>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 13 mins</html>

Chromosomal DNA isolation from E. coli protocol

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